Application of Meso Scale Technology for the Measurement of Phosphoproteins in Human Tumor Xenografts

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The serine/threonine kinase AKT plays a pivotal role in signal transduction events involved in malignant transformation and chemoresistance and is an attractive target for the development of cancer therapeutics. Fragment-based lead discovery, combined with structure-based drug design, has recently identified AT7867 as a novel and potent inhibitor of both AKT and the downstream kinase p70 S6 kinase (p70S6K) and also of protein kinase A. This ATP-competitive small molecule potently inhibits both AKT and p70S6K activity at the cellular level, as measured by inhibition of GSK3β and S6 ribosomal protein phosphorylation, and also causes growth inhibition in a range of human cancer cell lines as a single agent. Induction of apoptosis was detected by multiple methods in tumor cells following AT7867 treatment. Administration of AT7867 (90 mg/kg p.o. or 20 mg/kg i.p.) to athymic mice implanted with the PTEN-deficient U87MG human glioblastoma xenograft model caused inhibition of phosphorylation of downstream substrates of both AKT and p70S6K and induction of apoptosis, confirming the observations made in vitro. These doses of AT7867 also resulted in inhibition of human tumor growth in PTEN-deficient xenograft models. These data suggest that the novel strategy of AKT and p70S6K blockade may have therapeutic value and supports further evaluation of AT7867 as a singleagent anticancer strategy.

Section: Methodsmentioning

confidence: 99%

AT7867 Is a Potent and Oral Inhibitor of AKT and p70 S6 Kinase That Induces Pharmacodynamic Changes and Inhibits Human Tumor Xenograft Growth

Grimshaw

Hunter

Yap

et al. 2010

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“…IC 50 values were determined (30) 4 cells/mL in 96-well plates, and 48 h later, they were treated with compounds for 2 or 8 h. Medium was then removed, and 50 μL of lysis buffer was added. Plates were freeze thawed once at −80°C and 40 μL of lysate was transferred directly onto the Meso Scale Discovery plate, and analysis was completed as described previously (34). For each treatment condition, a single well from each of three independent plates was analyzed.…”

Section: Compound Supplymentioning

confidence: 99%

“…For dedicated pharmacodynamic studies, animals were dosed for 4 d and samples obtained as before. Plasma and tumor samples were analyzed for compound concentrations and tumor samples assessed for evidence of biomarker modulation by Meso Scale Discovery electrochemiluminescence immunoassay and/or immunoblot, as previously described (29,34). In some experiments, IGROV-1 human ovarian cancer xenografts were studied using similar methods to those for U87MG.…”

Section: Xenograft Tumor Efficacy and Pharmacodynamic Studiesmentioning

confidence: 99%

Biological properties of potent inhibitors of class I phosphatidylinositide 3-kinases: from PI-103 through PI-540, PI-620 to the oral agent GDC-0941

Raynaud

Eccles

Patel

et al. 2009

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248

226

The phosphatidylinositide 3-kinase pathway is frequently deregulated in human cancers and inhibitors offer considerable therapeutic potential. We previously described the promising tricyclic pyridofuropyrimidine lead and chemical tool compound PI-103. We now report the properties of the pharmaceutically optimized bicyclic thienopyrimidine derivatives PI-540 and PI-620 and the resulting clinical development candidate GDC-0941. All four compounds inhibited phosphatidylinositide 3-kinase p110α with IC 50 ≤ 10 nmol/L. Despite some differences in isoform selectivity, these agents exhibited similar in vitro antiproliferative properties to PI-103 in a panel of human cancer cell lines, with submicromolar potency in PTEN-negative U87MG human glioblastoma cells and comparable phosphatidylinositide 3-kinase pathway modulation. PI-540 and PI-620 exhibited improvements in solubility and metabolism with high tissue distribution in mice. Both compounds gave improved antitumor efficacy over PI-103, following i.p. dosing in U87MG glioblastoma tumor xenografts in athymic mice, with treated/control values of 34% (66% inhibition) and 27% (73% inhibition) for PI-540 (50 mg/ kg b.i.d.) and PI-620 (25 mg/kg b.i.d.), respectively. GDC-0941 showed comparable in vitro antitumor activity to PI-103, PI-540, and PI-620 and exhibited 78% oral bioavailability in mice, with tumor exposure above 50% antiproliferative concentrations for >8 hours following 150 mg/kg p.o. and sustained phosphatidylinositide 3-kinase pathway inhibition. These properties led to excellent dose-dependent oral antitumor activity, with daily p.o. dosing at 150 mg/kg achieving 98% and 80% growth inhibition of U87MG glioblastoma and IGROV-1 ovarian cancer xenografts, respectively. Together, these data support the development of GDC-0941 as a potent, orally bioavailable inhibitor of phosphatidylinositide 3-kinase. GDC-0941 has recently entered phase I clinical trials.

“…Tumor samples were homogenized using a buffer containing 50 mmol/L Tris (pH 7.4), 1 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton X-100, 1 mmol/L NaF, 1 mmol/L NaVO 4 , 5 μmol/L Fenvalerate, 5 μmol/L Vbphen, 10 mg/mL TLCK, 1× Complete inhibitor tablet per 10-mL buffer (Roche), protease inhibitor cocktail, and phosphatase inhibitor 1 and 2 (SigmaAldrich; ref. 33). Protein concentrations were measured by Bradford assay.…”

Section: Immunoblottingmentioning

confidence: 99%

The Preclinical Pharmacology and Therapeutic Activity of the Novel CHK1 Inhibitor SAR-020106

Walton

Eve

Hayes

et al. 2010

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Genotoxic antitumor agents continue to be the mainstay of current cancer chemotherapy. These drugs cause DNA damage and activate numerous cell cycle checkpoints facilitating DNA repair and the maintenance of genomic integrity. Most human tumors lack functional p53 and consequently have compromised G 1 -S checkpoint control. This has led to the hypothesis that S and G 2 -M checkpoint abrogation may selectively enhance genotoxic cell killing in a p53-deficient background, as normal cells would be rescued at the G 1 -S checkpoint. CHK1 is a serine/threonine kinase associated with DNA damage-linked S and G 2 -M checkpoint control. SAR-020106 is an ATP-competitive, potent, and selective CHK1 inhibitor with an IC 50 of 13.3 nmol/L on the isolated human enzyme. This compound abrogates an etoposide-induced G 2 arrest with an IC 50 of 55 nmol/L in HT29 cells, and significantly enhances the cell killing of gemcitabine and SN38 by 3.0-to 29-fold in several colon tumor lines in vitro and in a p53-dependent fashion. Biomarker studies have shown that SAR-020106 inhibits cytotoxic drug-induced autophosphorylation of CHK1 at S296 and blocks the phosphorylation of CDK1 at Y15 in a dose-dependent fashion both in vitro and in vivo. Cytotoxic drug combinations were associated with increased γH2AX and poly ADP ribose polymerase cleavage consistent with the SAR-020106-enhanced DNA damage and tumor cell death. Irinotecan and gemcitabine antitumor activity was enhanced by SAR-020106 in vivo with minimal toxicity. SAR-020106 represents a novel class of CHK1 inhibitors that can enhance antitumor activity with selected anticancer drugs in vivo and may therefore have clinical utility.